Process for the manufacture of alkaline earth metal salts of adenosine polyphosphoric acids



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Patented Feb. 29, 1944' rnoonss FOR THE MANUFACTURE OF, AL I mum: EARTH METAL SALTS or ADENO-: A

SINE roLYrnosrnomohcms v Herbert Schwaneberg, Berliii fle1np elhof, Germany; vested in the Alien Property'cl'l'stodian It isknown from th'e' work o ffNeedham' and van He'ynigenwhic'h was published'in Nature volume 135 1935) 135285 585 andl 586', that adenosine triphosphoricjacid is formed bythe addition of 'phosphoglyceric acid andadenylic acid to niuscle' extract containing alkali, Working with muscle ext 'act is, however, too coniplicatedand expensive, in practice, 7 them'or so because the yields obtained are not satisfactory; I I It isalso known from the work of Ostern and 'j'I'ei'szekowec published in Hopp'e seylrs J Zeitschrift fiir' physiologische f Ch'e niefyolume 250 1(19 3 7) pages 155-151 that'adenosine, in the presence of inorganic phosphateja id under the action of;yeast, forms adeno i triphosphoric acid Drawing ApplicationMayM, 1949, Serial No. 1 336,304. In Germany August}, 1939 cci ims. (01.195-28) I i'idenosineas such or any coinpound'which can be split upfto form adenosine, such. as for example yeast nucleic acids, can be used as the-starting,

material in the new process asintheknownfproc ess. As the phosphatethere maybeused either inorgnic phosphate bufier'solutionsjofalso the known sugarphosplior'ic acid esters such as, for

' 'example,...the so-called Neuberg, Robison,. Co'ri,

. in addition to adenosine' 5 monophosphoric acid.

[11A, proc ss. 01 {this nature is described "and Qclaimejd'in U. S.PatentN0.2,174,475 When working 1 in accordance" withgjthis process, how.-

e jer,Iadenosine-5-monophospho ric acid is mainly obtained; sometimes it is mixed with someade nosinelpolyphosphoric'acid. r

'An'obje'ct of my present invention is to obtain, instead of the above-mentioned mixture (which mane triphosphoricacid .or' alsowith some ade- 'Einb'den, Harden and "Young" esters; phosph'oglyceric acid or. mixturesiot organic and inorganic phosphate can also be.. used. These starting materials are convertedjby' the action of fresh yeast, dry yeast or yeast extractoryeast plasmolysate into adenosinetriphosphoric acid and v adenosine polyphosphoric acid. It is important that the yeast be addedto'thereaction -mi rt1ire in prefermented condition and {preferably in stages. The yeastcontains twoenzyrnes oiwhich the one, namely the so-called phosphatase, has a dephosphorylating action, 7 while simultaneously a second enzyme which isypresent in the Iyeast always. contains considerable amounts of adenosine monophosphoric acid," substantially only adenosine ,polypho'sphori'c acids, and", especially 'adenosine. triphosphoric acid, which are 75-'100% free J from adenosine i monophosphoric acid. r

A furtherobject my inyentiojnis to obtain a ,new wprep'a'ratio'n for acting on the circulatory has a phosphorylating, action,v namely m h'e ,5-

positionlof the ribose containedadenosine. Ithas nowbeen foundthat by avigorousfermenstation oi thev yeast the result carilbe obtained that this second enzyme. st ron y'p dom t Y so that the phosphorylation of the vadenosijne Proceeds not only as far asmuscle adenylicacid system, especially'of human beings, which is more emc'aciofiis than the known adenos'ine monophosl phjori c 'acidwhich has hithertofbe'en used for this purpose, namely a'prepai'ation which consists Io'rfthe niost part or adenosinexpolyphosphoric i ds and especially adenosine triphosphoric A still furtherobject or] my invention isto ob-s;

. v taintherapeutically valuable sailtsfsuch as,-for example, calcium, salts of adenos'ine-polyphosphoric acid s,' directly from thereaction mixture whichcontains the crude adenosine" polypho'sphoric acidsand adenosine 'triphos'pho'r'ic acid in pa'rticf ular', that is to saywithoutforming other com;

m m th s :i

gFinally," et another obje'ctof'my invention is I "to convert the adenosine polyphdsphoric acids directly/or indirectly into salts of varioustherapeutically valuable metals." I

" 'Furth'er objects and'advantages of inventionwill be clear from the tollowingide- (adenosine-5-monophos phoric I acid) but .I up to the .desired adenosine polyphosphoricsacid. The predomination of the phosphory1atingg-hzyme ,may be ensured, in particular "by adding the vigorously fermenting yeast in stages to the-reaction mixture inorder ;to prevent; with certainty theproductionloi phosphatase As soon as anboiled in order to destroy the phosphatasepresent. This boiling must take placeas guickly'as 40v possible and preferably within-about ,1Q'-20 minwa in'orderiltof r en dw ils s j' thex b phosphoric acidsffo rmed." For the same reason the highly heated ll st, also -be. rai idly cooled downrsince otherwise dephosphorylati can occur y ydrolysis;

The solution, obtained :in this manner which '7 contains practically the whole tofi -theadenosine in the tormof polyphosphoric, acidsiisipreferably worked up. in the following rnanner. 1

, The liquor which isfreed form insoluble-matmy present This barium salt is then treated with dilute sulphuric acid in such a way that the whole of the barium is present as sulphate. The solution, which contains the free adenosine triphosphoric acid and adenosine polyphosphoric acid is rapidly separated from the precipitate by centrifuging glyceric acid can be taken. The further treatin order that the excess of acid may cause no harmful hydrolysis and is then poured into 5-10 times its amount of alcohol. By this means adenosine triphosphoric acid is separated as a white powder. It is practically free from inorganic phosphate and is contaminated only by a-small quantity of hexose diphosphoric acid from which it can easily be separated if this is desired.

Another method which leads directly to therapeutically useful salts of adenosine poLvphosphoric acids is to treat the solution, after boiling and filtering, with a concentrated solution of a cal cium salt, for example calcium chloride, in the calculated quantity and to add alcohol. In this way the calcium salts of adenosine polyphosphoric acids are precipitated and can be used directly as a medicament.

The adenosine polyphosphoric acids which are obtained in a degree of purity from 75-100% can be used for the manufacture of potassium. sodium, magnesium, manganese salts and so forth which are important as agents for affecting the circulatory system.

The invention will now be explaine in greater detailwiththe aid of the following examples.

I Example 1 the whole experiment. Theamount of inorganic phosphorus taken up is continuously checked and the experiment is broken ofl when phosphorus is no longer taken up. Then the solution is rapidly boiled and then immediately cooled down and filtered. The solution is made alkaline with caustic soda solution and is precipitated with barium acetate, The crude barium adenosine polyphosphate is centrifuged andwashed with water and alcohol.

If the crude phosphate, while being cooled, is stirred with the necessary quantity of dilute sulphuric acid and at once centrifuged and the solution poured into several times its volume of alcohol and stirred, a white flocculent product is obtained which consists substantially of crude adenosine polyphosphoric acids. This is at once filtered on and washed with alcohol and ether. It is very sensitive to moisture and must be dried at once in the desiccator.

' Example 2 again added. Instead of fructose diphosphoric acid the corresponding. quantity of phosphoment is carried out in the same way as in Example 1.

Example 3 1 kg. of beer yeast is stirred with 1 litre of 10% glucose solution and after the vigorous fermentation has begun 5 litres of 1% adenosine solution, 150 grams of the sodium salt of fructose-diphosphoric acid in ,3 litres of water, or the corresponding quantity of the sodium salt of phosphoalyceric acid. and 300 cc. of toluene are added. The mixture is stirred and is kept at a temperature of 37 C. After 30 minutes 1 kg. of beer yeast which is fermenting with 1 litre of 10% glucose solution is again added. The further treatment then proceeds as described in Example 2.

Some preferred methods of carrying the invention into effect have been described in detail, but the invention is not limited inany way to the reaction temperatures, concentrations and other conditions which have been numerically indicated. These can be varied according to the circumstances of a particular case without departing from the scope of the invention. The invention is limited only by the following claims, taking into account the state of the prior art.

I claim: I

1. A process for the manufacture of alkaline earth metal salts of adenosine polyphosphoric acids which comprises the followingsteps: attaching phosphoric acid residues to adenosine by the action of yeast by. introducing yeast in vigorous fermenting condition 'in batches into a reaction mixture containing adenosine, a sugar which is fermentable by yeast and a phosphoric acid residue, leaving theinixture to fermentation after the addition of yeast, rapidly boiling the mixture. then rapidly cooling it down, separating the soluble fromthe insoluble matter, adding an alkaline earth to the solution and separating the alkaline earth metal salts of the adenosine polyphosphoric acids. i

2. A process for the manufacture of adenosine I polyphosphoric acids comprising the following steps: attaching phosphoric acid residues to adenosine by introducing yeast in vigorous fermentating condition in a plurality of batches into a reaction mixture containing adenosine, a sugar which is fermentable by yeast andpolyphosphoric acid residues, leaving the mixture to fermentation after the addition of yeast, quickly boiling the solution, then rapidly cooling it down, separating the soluble from the insoluble matter, adding an alkaline earth to the solution obtained, separating the alkaline earth metal salts of adenosine polyphosphoric acids, decomposing these salts with a mineral acid and precipitating by addition of alcohol the adenosine polyphosphoric acids from the solution.

3. A process for the manufacture of salts of I adenosine polyphosphoric acids comprising the following steps: attaching phosphoric acid residues to adenosine by the action of yeast by introducing vigorously fermenting yeast in a plurality of batches into a reaction mixture containing adenosine, a sugar which is fermentable by yeast and-phosphoric acid residues, leaving the mixture to fermentation after the addition of yeast, rapidly boiling the mixture, then rapidly cooling it down, adding an alkaline earth to the solution obtained, separating the alkaline earth metal salts thereby obtained of the adenosine polyphosphoric acids, decomposing the salts obtained with acid, precipitating adenosine polyphosphoric attaching phosphoric acid residues to yeast in prefermented condition in aplurality of batches into a reaction mixture containing adenosine, a sugar which is fermentable by yeast, and phosphoric acid residues leaving the mixture to fermentation at about 37 'C. after the addition of yeast, rapidly boiling the mixture within a time which is not substantially longer than 20 minutes, then rapidly cooling it down, adding an alkaline earth, separating the alkaline earth metal salts thereby obtained of the adenosine polyphosphoric acids and decomposing these salts with mineral acids. 1

5. A process for the manufacture of adenosine polyphospnoric acids comprising the following steps: attaching phosphoric acid residues to adenosine by the action of yeast by adding a plurality of batches of vigorously fermenting yeast to a reaction mixture whichv contains adenosine, a

sugar which is fermentable by yeast and an organic phosphate buffer solution having a pH value of about 7, leaving the mixture to fermentation after the addition of yeast, rapidlyboiling the mixture, rapidly cooling it down, adding an alkaline earth to the solution obtained, separating the alkaline earth metal salts of the adenosine polyphosphoric acids and decomposing these salts with acid. v

6. A process for the manufacture of adenosine polyphosphoric acids comprising the following steps: attaching phosphoric acid residues to adenosine by the action of yeast by adding yeast in prefermented condition in several batches to a reaction mixture containing adenosine, a sugar which is fermentable by yeast and a sugar-phosphoric acid ester, leaving the mixture to fermentation after the addition of yeast, rapidly boiling the mixture, ihen rapidly cooling it down, adding an alkaline earth to the solution obtained, separating the alkaline earth metal salts of the ade tion of yeast by introducing yeast in prefermented a condition-and in a plurality of batches into a reaction mixture containing adenosine, a sugar which is fermentable by yeast, and phosphoric acid residues, rapidly boiling the mixture, rapidly phoric acid and decomposing these salts with acid.

' 9. A process as claimed in claim 8 whc nn the pH value of the reaction mixture containing the yeast, adenosine and phosphoric acid residue is kept at about 7- by the presence of an inorganic phosphate buffer solution.

HERBERT SCHWANEBERG. 

